Fundamental Characterization of Antibody Fusion-Single-Chain TNF Recombinant Proteins Directed against Costimulatory TNF Receptors Expressed by T-Lymphocytes

The costimulatory signal regulated by the members of the tumor necrosis factor receptor (TNFR) superfamily expressed by T cells plays essential roles for T cell responses and has emerged as a promising target for cancer immunotherapy. However, it is unclear how the difference in TNFR costimulation contributes to T cell responses. In this study, to clarify the functional significance of four different TNFRs, OX40, 4-1BB, CD27 and GITR, we prepared corresponding single-chain TNF ligand proteins (scTNFLs) connected to IgG Fc domain with beneficial characteristics, i.e., Fc−scOX40L, Fc−sc4-1BBL, Fc−scCD27L (CD70) and Fc−scGITRL. Without intentional cross-linking, these soluble Fc−scTNFL proteins bound to corresponding TNFRs induced NF-kB signaling and promoted proliferative and cytokine responses in CD4+ and CD8+ T cells with different dose-dependencies in vitro. Mice injected with one of the Fc−scTNFL proteins displayed significantly augmented delayed-type hypersensitivity responses, showing in vivo activity. The results demonstrate that each individual Fc−scTNFL protein provides a critical costimulatory signal and exhibits quantitatively distinct activity toward T cells. Our findings provide important insights into the TNFR costimulation that would be valuable for investigators conducting basic research in cancer immunology and also have implications for T cell-mediated immune regulation by designer TNFL proteins.

Our understanding of costimulatory TNFSF-TNFRSF interactions has progressed, and the wealth of experimental results are currently applied to the development of biologics or gene engineering technologies [15][16][17][18][19][20][21]. Despite rational strategies to generate biotherapeutics targeting the TNFSF-TNFRSF molecules, these efforts are frequently hindered by a lower performance in clinical trials [22][23][24]. We think that more fundamental analyses are still required to understand the role of costimulatory TNFSF-TNFRSF interactions in T cell responses. Unlike antagonists, where the function of molecules is basically to block the receptor-ligand interaction, the action of agonists is more diverse and their regulatory mechanisms are difficult to generalize. Thus, it is important to attain basic information on how costimulatory TNFRSF molecules exhibit their activities through interaction with agonists and what is the relative contribution of each TNFRSF molecule to others in regulating T cell responses. We believe that deciphering these questions would be important for the future development of novel TNFR-targeted immunotherapies.
In the present study, we showed that these four Fc−scTNFL proteins bound to CD4 + and CD8 + T cells, which led to the induction of cell proliferation and cytokine release with different dose dependencies. Fc−scGITRL induced a potent T cell recall response in vivo. The fundamental information obtained in this study provides important insights into TNFR costimulation that would be helpful for investigators conducting fundamental immunological research and has implications for T cell-mediated immune regulation by engineered TNF proteins.
Genes for sc4-1BBL, scCD70 and scGITRL were constructed as follows. Three PCR fragments containing an identical nucleotide sequence corresponding to an extracellular THD region of 4-1bb ( 122 Arg− 309 Glu), Cd70 ( 45 Ser− 195 Pro) or Gitr ( 42 Thr− 173 Ser) were amplified using three different primer pair sets. The first PCR fragment was amplified with a forward primer containing a 5 -KpnI site and a reverse primer containing a 3 -BamHI site within the GGGSGGG linker. The second PCR fragment was amplified with a forward primer containing a 5 -BamHI site within the GGGSGGG linker (ggt gga gga tcc ggt gga ggt) and a reverse primer containing a 3 -BspEI site within the GGGSGGG linker. The third PCR fragment was amplified with a forward primer containing a 5 -BspEI site within the GGGSGGG linker (gga ggc ggt tcc gga ggt ggg) and a reverse primer containing a 3 -AgeI site. The gel purified three PCR fragments which were digested with BamHI (R3136, New England Biolabs) and BspEI (R0540, New England Biolabs) were connected to each other using Ligation High TM (LGK-101, TOYOBO, Osaka, Japan). After amplification with PCR, the entire gene for sc4-1BBL, scCD70 or scGITRL, (KpnI)−THD−GGGSGGG−THD−GGGSGGG−THD−(AgeI), was ligated into qCR TM -Blunt II-TOPO TM . The pCAGGS vector encoding Fc−PA−sc4-1BBL−His 6 was constructed by replacing the gene for scOX40L in the pCAGGS vector encoding Fc−PA−scOX40L−His 6 with the gene for sc4-1BBL in qCR TM -Blunt II-TOPO TM vector, after enzymatic digestion with KpnI (R3142, New England Biolabs) and AgeI (R3552, New England Biolabs) ( Figure  S1B,C). The pCAGGS vector encoding Fc−PA−scCD70−His 6 or Fc−PA−scGITRL−His 6 was constructed in the same manner described above ( Figure S1D,E).
A plasmid encoding mouse OX40 extracellular region ( 21 Thr-211 Pro) and human IgG1-Fc (mouse OX40-Fc) in the pEF-Fc vector [41], and a plasmid encoding mouse 4-1BB extracellular region ( 24 Val− 187 Leu) and human IgG1-Fc (mouse 4-1BB-Fc) in the pEF-Fc vector [37] were previously described. A plasmid encoding mouse CD27 extracellular region ( 24 Pro− 182 Arg) and human IgG1-Fc (mouse CD27-Fc) in the pEF-Fc vector was constructed in this study, and a PCR fragment (gac tac aag gat gac gat gac aag ctc gat gga gga tac cca tac gat gtt cca gat tac gct) corresponding to a Flag−HA tag peptide (DYKDDDDKLDGGYPY-DVPDYA) was inserted into the 3 end of the CD27−Fc gene by In-Fusion cloning.
Fc−scTNFL proteins were produced with average yield of 10 to 100 mg per liter in the culture supernatant of stably transfected CHO cells.

ELISA
For screening of cell clones producing higher levels of Fc−scTNFL proteins, culture supernatants were added to ELISA plates (439454, Thermo Fisher, Waltham, MA, USA) precoated with 0.5 µg/mL of mouse anti-His tag IgG (011-23091, FUJIFILM), and the binding between anti-His tag IgG and Fc−scTNFL was visualized by 0.07 µg/mL of biotin-anti-human IgG and HRP-streptavidin (405210, BioLegend) at a 1:3000 dilution.

Delayed-Type Hypersensitivity Response
Delayed-type hypersensitivity (DTH) response was evaluated as previously described [37,42]. C57BL/6 mice from both male and female mice aged between 12 and 14 weeks were immunized subcutaneously at the tail with 200 µL of 1.25 mg/mL methyl BSA (mBSA) (A1009, Merck) emulsified with complete Freund's adjuvant (CFA) (F5881, Merck) on day 0. Seven days after the immunization, mice were challenged subcutaneously in a footpad with 30 µL of 7 mg/mL mBSA in PBS plus 10 µL of PBS or 1000 µg/mL of Fc−scGITRL. An equal volume of PBS was injected into another footpad as a control. One day after the challenge, footpad thickness was measured with a digital caliper (Shinwa Rules Co., Ltd., Japan). The magnitude of the DTH response was determined as follows: [footpad swelling (%)] = ([footpad thickness of mBSA-injected footpad (mm)] − [footpad thickness of PBS-injected footpad (mm)]) ÷ [footpad thickness of PBS-injected footpad (mm)] × 100. Each frozen block of footpad was cut into 5-µm-thick sections. After fixing with 100% ethanol and 4% paraformaldehyde, samples were stained with hematoxylin and eosin (H&E). Images of each footpad section were taken using a fluorescence microscope (BZ-X800, Keyence, Osaka, Japan).

Statistical Analysis
Statistical significance was determined by a two-tailed unpaired Student's t test for two groups, with the assumption of normal distribution of data and equal sample variance. A p value of less than 0.05 was considered statistically significant.

Specific Binding of Fc−scTNFL Fusion Protein to Corresponding TNFR on T Cells
TNFRSF molecules on T cells and TNFSF molecules on APCs interact during the course of immune responses, and these physiologically relevant TNFRSF-TNFSF interactions regulate immunity and diseases. The activation of TNFRSF molecules plays important roles in the control of T cell immunity, and thus it is very important to understand what types of molecules can be a substitute for naturally expressing TNFSF molecules on APCs. The information would be beneficial for the design of novel therapeutic agents for infections and cancers.
To understand how the activity of TNFRSF molecules expressed by CD4 + and CD8 + T cells is controlled by engineered TNFSF molecules, we prepared four scTNFL proteins, scOX40L, sc4-1BBL, scCD70 and scGITRL, in which three identical THD protomers were covalently connected to each other with two GGGSGGG peptide linkers to support the organization of the trimer structure of TNF ligand protein. For the convenience of protein detection and purification, a PA-peptide tag [45] and a His 6 -peptide tag were attached to both ends of the scTNFL, and additionally, IgG Fc domain was connected to the Nterminus of the PA−scTNFL−His 6 to create Fc−scTNFL fusion proteins, i.e., Fc−scOX40L, Fc−sc4-1BBL, Fc−scCD70 and Fc−scGITRL ( Figure 1). These four Fc−scTNFL proteins purified from culture supernatants displayed a SS-linked dimer structure in SDS-PAGE ( Figure 2). Thus, one Fc−scTNFL molecule has two TNFL trimer units and forms a hexameric structure.
Based on the expression profile described above, we firstly confirmed the levels of OX40, 4-1BB, CD27 and GITR on CD4 + and CD8 + T cells using staining antibodies for these TNFRs before and after T cell activation. The expression of OX40 and 4-1BB on CD4 + and CD8 + T cells was upregulated after T cell activation ( Figure 4A,C). In contrast, CD27 was highly expressed by unstimulated CD4 + and CD8 + T cells, and the expression was greatly downregulated after T cell activation ( Figure 4A,C). GITR was also expressed by TNFRs before and after T cell activation. The expression of OX40 a CD8 + T cells was upregulated after T cell activation ( Figure 4A,C). highly expressed by unstimulated CD4 + and CD8 + T cells, and the e downregulated after T cell activation ( Figure 4A,C). GITR w unstimulated CD4 + and CD8 + T cells, and the expression was main after T cell activation ( Figure 4A,C). Consistent with the above results, all four Fc−scTNFL proteins bound to the surface of CD4 + and CD8 + T cells ( Figure 4B,D), and the staining profile of Fc−scTNFL proteins was comparable with that of staining antibodies ( Figure 4A-D).
Collectively, these results demonstrate that all four Fc−scTNFL proteins consistently exhibit specific binding activity toward corresponding TNFRs expressed by CD4 + and CD8 + T cells.

NF-kB Activation by Fc−scTNFL
The classical NF-kB pathway plays essential roles in T cell activation. The degradation of IkBa protein has been used as a representative marker for the activation of the classical NF-kB signaling. Costimulatory TNFRs including OX40, 4-1BB, CD27 and GITR are major activators of the NF-kB pathway [1,55,56]. Our group previously established a T cell hybridoma cell line expressing OX40 [36,43,44]. This cell also endogenously expressed CD27 and GITR at a steady state ( Figure 5A), and 4-1BB could be induced after stimulation with PMA and ionomycin ( Figure 5A,C). The binding profile of Fc−scTNFL proteins was comparable with that of antibodies for TNFRs ( Figure 5A). To evaluate the NF-kB pathway activation, this T cell was cultured with respective Fc−scTNFL proteins. and CD8 (C,D) T cells were stained with indicated antibodies for TNFRs (A,C) or with indicated Fc−scTNFL proteins (B,D), evaluated by flow cytometry. CD4 + or CD8 + T cells were stained right after purification (before stimulation) or 3 days after stimulation with anti-CD3 and -CD28 antibodies plus IL-2 (after stimulation). Positive staining is shown by white histogram. Negative staining control is shown by shaded histogram. Data are from one experiment representative of at least two independent experiments with similar results. Consistent with the above results, all four Fc−scTNFL proteins bound to the surface of CD4 + and CD8 + T cells ( Figure 4B,D), and the staining profile of Fc−scTNFL proteins was comparable with that of staining antibodies ( Figure 4A-D).
Collectively, these results demonstrate that all four Fc−scTNFL proteins consistently exhibit specific binding activity toward corresponding TNFRs expressed by CD4 + and CD8 + T cells.

NF-kB Activation by Fc−scTNFL
The classical NF-kB pathway plays essential roles in T cell activation. The degradation of IkBa protein has been used as a representative marker for the activation of the classical NF-kB signaling. Costimulatory TNFRs including OX40, 4-1BB, CD27 and GITR are major activators of the NF-kB pathway [1,55,56]. Our group previously established a T cell hybridoma cell line expressing OX40 [36,43,44]. This cell also endogenously expressed CD27 and GITR at a steady state ( Figure 5A), and 4-1BB could be induced after stimulation with PMA and ionomycin ( Figure 5A,C). The binding profile of Fc−scTNFL proteins was comparable with that of antibodies for TNFRs ( Figure 5A). To evaluate the NF-kB pathway activation, this T cell was cultured with respective Fc−scTNFL proteins. Upon stimulation with soluble Fc−scOX40L, IkBa was degraded in a time-dependent manner in this T cell ( Figure 5B). Similarly, stimulation of T cells with Fc−scCD70 or Fc−scGITRL resulted in IkBa degradation ( Figure 5B), and Fc−sc4-1BBL induced the degradation of IkBa in activated T cells ( Figure 5C,D). Thus, all Fc−scTNFL proteins activate the classical NF-kB pathway mediated by OX40, 4-1BB, CD27 and GITR.
To evaluate costimulatory activity of Fc−scTNFL proteins, CD4 + or CD8 + T cells purified from mouse spleens were stimulated with a fixed concentration (10 µg/mL) of platebound anti-CD3 agonistic antibody and increasing concentrations of soluble Fc−scTNFL proteins. Upon stimulation of CD4 + T cells with anti-CD3, additional Fc−scOX40L significantly promoted IL-2 and IFN-g production and cell proliferation ( Figure 6A). Similarly, Fc−sc4-1BBL, Fc−scCD70 or Fc−scGITRL dose-dependently induced cytokine release and proliferation of CD4 + T cells (Figure 6A), showing a potent costimulatory activity of Fc−scTNFL proteins for CD4 + T cells. Notably, Fc−scGITRL could efficiently increase IL-2 from CD4 + T cells even at 0.3 µg/mL ( Figure 6A). As with CD4 + T cells, Fc−sc4-1BBL, Fc−scCD70 and Fc−scGITRL also significantly augmented cytokine and proliferative responses of CD8 + T cells ( Figure 6B). In contrast, Fc−scOX40L displayed a less potent activity for CD8 + T cells, as compared to CD4 + T cells. Interestingly, Fc−sc4-1BBL could efficiently promote the production of IFN-g and proliferation, as compared to other Fc−scTNFL proteins ( Figure 6B). Thus, these results demonstrate that Fc−scTNFL proteins may have redundant and specific functions in promoting costimulatory signaling pathways in CD4 + and CD8 + T cells.
To understand how the costimulatory signal mediated by Fc−scTNFL proteins is dependent on the TCR/CD3 signal, CD4 + or CD8 + T cells were stimulated with increasing concentrations of anti-CD3 in the presence or absence of a fixed concentration (1 µg/mL) of respective Fc−scTNFL proteins. This analysis revealed that higher concentrations of anti-CD3 were required for Fc−scTNFL proteins to increase cytokine production and cell proliferation in both CD4 + and CD8 + T cells ( Figure 7A,B). Notably, Fc−scGITRL exhibited better costimulatory activity against CD4 + T cells as compared to other Fc−scTNFL proteins ( Figure 7A). Fc−scGITRL induced the highest amount of IL-2 from CD4 + T cells ( Figure 7A), whereas Fc−sc4-1BBL produced the largest amount of IFN-g from CD8 + T cells ( Figure 7B).
These results show that all four Fc−scTNFL proteins retain an active TNF ligand structure to be able to agonize corresponding TNFRs expressed by CD4 + and CD8 + T cells and efficiently promote the production of cytokines and proliferation in a dosedependent manner. To understand how the costimulatory signal mediated by Fc−scTNFL proteins is dependent on the TCR/CD3 signal, CD4 + or CD8 + T cells were stimulated with increasing concentrations of anti-CD3 in the presence or absence of a fixed concentration (1 µg/mL) of respective Fc−scTNFL proteins. This analysis revealed that higher concentrations of ( Figure 7A), whereas Fc−sc4-1BBL produced the largest amount of IFN-g from CD8 T cells ( Figure 7B).
These results show that all four Fc−scTNFL proteins retain an active TNF ligand structure to be able to agonize corresponding TNFRs expressed by CD4 + and CD8 + T cells and efficiently promote the production of cytokines and proliferation in a dose-dependent manner.

DTH Response Mediated by Fc−scGITRL
To demonstrate costimulatory activity of Fc−scTNFL proteins in vivo, mice were immunized with mBSA on day 0 and challenged with mBSA with or without Fc−scTNFL on day 7 to induce DTH response, classified as type IV hypersensitivity associated with CD4 + T cell inflammation, as determined by footpad swelling on day 8. In our preliminary experiments, injection of one of the Fc−scTNFL proteins, Fc−scGITRL, in the challenge phase showed a trend toward increased DTH response, as compared to other Fc−scTNFL proteins. We thought that this might be related to the higher costimulatory activity of Fc−scGITRL for CD4 + T cells as shown in Figures 6A and 7A. For this reason, we decided to carefully examine whether Fc−scGITRL could impact the DTH response.
The footpad swelling was significantly enhanced in mice injected with mBSA plus Fc−scGITRL (G2) compared to mBSA alone (G1) within 1 day after the challenge ( Figure 8A,B). Fc−scGITRL induced the increment in the epidermal thickness of footpad skin (Figures 8C  and S2). Spleen cells from mice challenged with mBSA and Fc−scGITRL (G2) showed significantly higher proliferative responses to mBSA as compared to those with mBSA alone (G1) ( Figure 8D). These results indicate that Fc−scGITRL rapidly augments T cell-dependent inflammation in the challenge phase of DTH response.
independent experiments with similar results. * p < 0.05 for comparison between with and withou Fc-scTNFL in respective anti-CD3 concentrations (Student t test).

DTH Response Mediated by Fc−scGITRL
To demonstrate costimulatory activity of Fc−scTNFL proteins in vivo, mice wer immunized with mBSA on day 0 and challenged with mBSA with or without Fc−scTNF on day 7 to induce DTH response, classified as type IV hypersensitivity associated wit CD4 + T cell inflammation, as determined by footpad swelling on day 8. In our preliminar experiments, injection of one of the Fc−scTNFL proteins, Fc−scGITRL, in the challeng phase showed a trend toward increased DTH response, as compared to other Fc−scTNF proteins. We thought that this might be related to the higher costimulatory activity o Fc−scGITRL for CD4 + T cells as shown in Figures 6A and 7A. For this reason, we decide to carefully examine whether Fc−scGITRL could impact the DTH response.
The footpad swelling was significantly enhanced in mice injected with mBSA plu Fc−scGITRL (G2) compared to mBSA alone (G1) within 1 day after the challenge ( Figur  8A,B). Fc−scGITRL induced the increment in the epidermal thickness of footpad ski (Figures 8C and S2). Spleen cells from mice challenged with mBSA and Fc−scGITRL (G2 showed significantly higher proliferative responses to mBSA as compared to those wit mBSA alone (G1) ( Figure 8D). These results indicate that Fc−scGITRL rapidly augments cell-dependent inflammation in the challenge phase of DTH response.
Taken together, the results obtained in this study demonstrate that Fc−scTNF proteins efficiently enhance CD4 + and CD8 + T cell activation with different dose dependencies and suggest that a rational arrangement of TNFR agonism would fulfi roles in promoting the activation, differentiation and survival of antigen-primed T cells.  Taken together, the results obtained in this study demonstrate that Fc−scTNFL proteins efficiently enhance CD4 + and CD8 + T cell activation with different dose-dependencies and suggest that a rational arrangement of TNFR agonism would fulfill roles in promoting the activation, differentiation and survival of antigen-primed T cells.

Discussion
Over the past few decades, the TNFSF-TNFRSF of costimulatory molecules have been investigated to understand the important attributes of the T cell response, and especially the cognate interactions of OX40L-OX40, 4-1BBL-4-1BB, CD70-CD27 and GITRL-GITR have been shown to be critical for immune regulation by T cells. The aim of this study is to clarify the functional significance of four different TNFRSF molecules, OX40, 4-1BB, CD27 and GITR, in the regulation of the TNFR costimulation by using recombinant Fc−scTNFL proteins, i.e., Fc−scOX40L, Fc−sc4-1BBL, Fc−scCD70 and Fc−scGITRL. Without intentional cross-linking, soluble Fc−scTNFL proteins concurrent with anti-CD3 induced significant cytokine production and cell proliferation, as compared with anti-CD3 alone, showing that Fc-scTNFL proteins provide a potent costimulatory signal to T cells. The results demonstrate differential costimulatory activity of the scTNFL moiety of the fusion molecule and suggest that each individual cognate interaction quantitatively contributes to the TNFR costimulation that is essential for T cell activation. Our findings provide novel insights into the TNFSF-TNFRSF of costimulatory molecules in T cell responses and have important implications for the control of immune mediated diseases, including infections and cancers.
It has been a matter of concern how OX40, 4-1BB, CD27 and GITR regulate T cell responses. Expression levels of these TNFRs and their ligands are differentially controlled by the activation status of T cells and APCs, and these TNFRs might use different combinations of TNFR-associated factor (TRAF) family molecules to activate downstream signaling pathways. Thus, these costimulatory TNFRs may work cooperatively and/or independently. However, the functional significance of each individual TNFR for the activation of T cells has not been fully understood.
To clarify costimulatory activity of OX40, 4-1BB, CD27 and GITR, we decided to prepare corresponding Fc-scTNFL proteins, Fc−scOX40L, Fc−sc4-1BBL, Fc−scCD70 and Fc−scGITRL, respectively. For construction of the Fc−scTNFL gene, three copies of the extracellular THD genes were connected with two flexible glycine-serine-linker (GGGSGGG) genes, followed by linking to the C-terminus of the gene for the Fc fragment of human IgG1. The concatenated gene of Fc-scTNFL was inserted into the expression plasmid pCAGGS. The genetically engineered Fc-scTNFL proteins were purified from culture supernatants of CHO cells, followed by affinity chromatography purification with Protein A column. Accurate Fc dimer assembly of the Fc-scTNFL protein was confirmed by SDS-PAGE, indicating formation of a hexameric TNFL structure. All Fc-scTNFL proteins specifically interacted with their corresponding TNFRSF molecules and activated the NF-kB pathway, indicating the formation of an active TNF trimer structure. Finally, we verified that all four Fc-scTNFL proteins agonize the TNFRs expressed by CD4 + and CD8 + T cells. Our preliminary results suggest that the hexameric Fc-scTNFL molecule with two trimeric TNFL subdomains displayed more potent agonistic activity than one trimeric TNFL molecule (unpublished observation). Thus, the molecular format of scTNFL and IgG Fc domain would be generally beneficial for creating an agonist for TNFRSF molecules.
The expression levels of TNFRs on the surface of T cells are primarily important for the costimulatory function of TNFRs. It has been reported that OX40 expression on CD4 + and CD8 + T cells is enhanced by increasing engagement of the TCR/CD3 and that under physiological conditions, other costimulatory signals, including CD28, augment the expression of OX40. IL-1, IL-2, IL-4 and TNF-a also contribute to the prolonged expression of OX40. In this study, for the activation of CD4 + and CD8 + T cells in vitro we did not add anti-CD28 agonistic antibody in the culture (we added anti-CD28 antibody in the stimulation culture to optimally induce the upregulation of OX40 in the experiment of Figure 4). The lower concentration of anti-CD3 might not support the expression of OX40 in this in vitro culture setting. This may be the reason why we could only see increased cytokine and proliferative responses mediated by Fc-scOX40L at 10 µg/mL of anti-CD3 condition in Figure 7. A critical question would be how the expression level of TNFRSF molecules on T cells correlates with the activity of T cells. In the physiological setting, this might be influenced by the availability of antigens and cognate TNF ligands on APCs and by downregulation or shedding of TNF receptors in the case of CD27 [57,58]. It will be important to elucidate how the expression levels of costimulatory TNFRs have an impact in the T cell responses induced by Fc-TNFL proteins in more physiologically relevant conditions.
In this study, we could confirm a critical attribute of the 4-1BB costimulation for the activation of CD8 + T cells in terms of proliferation and cytokine production. Using cell lines expressing anti-CD3 antibody and membrane-bound TNFSF molecules, Kober et al. [59] reported that 4-1BBL showed the most potent activity for CD8 + T cells, as compared to OX40L, CD70, GITRL, CD30L or LIGHT. Using plate-immobilized HLApeptide complex and TNFSF molecules, Neuyen et al. [57] examined the role of the TNFR costimulation in CD8 + T cells and demonstrated that the 4-1BB costimulation showed the strongest amplification of cytokine production, as compared to CD27, GITR or OX40. Warwas et al. [60] tested costimulatory activities of TNFSF molecules using bifunctional recombinant fusion proteins and demonstrated that 4-1BBL exhibited a better costimulatory capacity than OX40L or CD70. These studies show that the 4-1BBL−41BB interaction plays a more predominant role for the activation of CD8 + T cells.
Additionally, we characterized the functional significance of the GITR costimulation for the activation T cells. Although Kober et al. [59] reported that costimulatory function of cellassociated GITRL was significantly lower than 4-1BBL, CD70 or OX40L, engineered GITRL fusion proteins efficiently promote T cell responses [61][62][63][64]. We found that Fc−scGITRL stimulated a greater production of IL-2 from CD4 + T cells. Administration of Fc−scGITRL concurrently with antigen into mice in the challenge phase significantly promoted the DTH response as determined by footpad swelling, implying that the GITR costimulation critically augments recall T cell responses. Thus, it will be important to elucidate how respective Fc−scTNFL proteins control different types of T cell responses in vivo in future studies.
Introduction of costimulatory intracellular domains from TNFRSF molecules, including OX40, 4-1BB, CD27 and GITR, into chimeric antigen receptor (CAR) constructs for CAR-T cell therapy has been demonstrated to effectively improve the functionality of CAR-T cells, i.e., T cell longevity and antitumor effects [65]. Among the four TNFRSF molecules focused on in this study, the intracellular costimulatory domain of 4-1BB has been the most extensively studied. Second-generation CARs incorporating either 4-1BB or CD28 costimulatory domain have proven to be effective and have been widely used in the clinic. The 4-1BB domain is advantageous over the CD28 domain in terms of inducing beneficial activity of CAR-T cells, which might be explained by the enhanced mitochondrial biogenesis and oxidative metabolism [66]. Although characterization of CARs with costimulatory domains of OX40, CD27 and GITR is lagging behind in comparison to CARs with 4-1BB, these TNFRSF domains have been demonstrated to be potent CAR-T cell drivers [67][68][69][70][71]. Further research regarding rational selection and engineering of intracellular TNFRSF domains would be important to optimize CAR-T activity that leads to therapeutic benefit.
The interplay among the TNFSF-TNFRSF of costimulatory molecules has been suggested to play a vital role for immune regulation, and a rational arrangement of costimulatory signals via OX40, 4-1BB, CD27 and GITR might be important for establishing protective T cell-immunity for virus infections. Choi et al. [72] reported that B cells expressing Epstein-Barr virus (EBV) signaling molecule LMP1 had higher levels of CD70, OX40L and 4-1BBL on the cell surface and that the costimulatory signal mediated by these TNFSF molecules induced potent cytotoxic CD4 + and CD8 + T cell responses. This study suggests that CD70, OX40L and 4-1BBL on antigen-presenting B cells promote the differentiation and expansion of antigen-specific cytotoxic T cells that eradicate EBV-related cancers. In accordance with this, immune responses against EBV infection are compromised in patients with inborn errors of CD70, CD27, TNFSF9 (4-1BBL) or TNFRSF9 (4-1BB) [73][74][75][76][77][78]. The CD27 costimulation in CD8 + T cells initially promoted the expression of 4-1BB and subsequent activation of the 4-1BB costiumlation [57]. Concurrent activation of OX40 and 4-1BB pathways was effective for inducing cytotoxic CD4 + Th1 cells with tumoricidal function [79]. These studies suggest that an amalgam of signals via TNFRs augments and prolongs the duration of protective T cell responses. If this is correct, combination treatment of TNF ligand proteins may be advantageous for control of EBV infection and related cancers.
In summary, we demonstrated that soluble Fc−scTNFL proteins efficiently activated CD4 + and CD8 + T cells with different dose-dependencies. The results have implications for immune regulation by rationally engineered TNF ligand molecules. Cell surface presentation of scTNFL protein moieties might be a better way to control T cell-mediated immunity. A scTNFL domain as the antigen-binding domain of CAR was used for cancer immunotherapy [80]. If selected sets of scTNFLs are efficiently presented on the surface of APCs, these cells would be beneficial for the induction of antigen-specific immune responses. Fc-scTNFL molecules using in this study can be humanized, and the human scT-NFL moiety presented on the surface of T cells or APCs might promote immune-mediated tumor destruction in a human setting. Further research will provide additional information regarding TNFR-targeted T cell regulation that may be advantageous for therapeutic intervention for infections and cancers.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author T.S. upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.